Uses of lipopolysaccharide binding protein

ABSTRACT

Novel LBP compositions and therapeutic uses for LBP are provided for preventing the adverse effects of exposure to endotoxin.

This is a Continuation of U.S. application Ser. No. 08/955,660, filedOct. 22, 1997 now U.S. Pat. No. 5,990,082.

The present invention relates to a novel use of lipopolysaccharidebinding protein (LBP) as a prophylactic/therapeutic agent in blockingthe pathological effects of lipopolysaccharide (LPS), also known asendotoxin.

BACKGROUND OF THE INVENTION

LPS is a major component of the outer membrane of gram-negative bacteriaand consists of serotype-specific O-side chain polysaccharide linked toa conserved region of core oligosaccharide and lipid A. LPS is a potentinducer of inflammation, stimulating the expression of manypro-inflammatory and pro-coagulant mediators in monocytes, macrophagesand endothelial cells. These responses are important in containing andeliminating a localized infection, however, adverse effects of systemicexposure to LPS can include induction of an inflammatory cascade, damageto endothelium, widespread coagulopathies, and organ damage. Systemicexposure to LPS can arise from direct infection of gram negativebacteria, leading to the complications of gram-negative sepsis [Traceyet al., Adv. Surg. 23: 21-56 (1990)]. Alternatively, a variety ofconditions and circumstances, including trauma, can induce changes ingut permeability that result in translocation of bacteria, and thereforeLPS, into circulating blood. Bacterial LPS translocated from the gut isthought to play a major role in post-surgical immunosuppression [Littleet al., Surgery 114(1): 87-91(1993)] and hemorrhagic shock. Therefore,there exists a need to discover therapies that can counteract theeffects of LPS in pathologic situations.

Two proteins, CD14 and lipopolysaccharide binding protein (LBP)[Schummann et al., Science 249: 1429-1431 (1990); Wright et al., Science249: 1431-1433 (1990)] have been shown to be required to generate aninflammatory response to LPS. LPS must bind to CD14 to activate aninflammatory response. CD14 is a 55 kD protein expressed via aglycosylphosphatidylinositol-anchor on the surface of macrophages,monocytes and neutrophils (mCD14). Endothelial and ephithelial cells,which do not express the CD14 protein, are activated by LPS bound to asoluble form of (sCD14) found in serum or plasma (at a concentration ofabout 2 μg/mL in normal human blood). CD14 preferentially binds to LPSmonomers [Tobias et al., J. Biol. Chem. 270(18): 10482-10488 (1995)].Since purified LPS exists in aqueous solution in micelles or aggregates,direct binding of LPS to CD14 is very slow [Tobias et al. (1995), supra;Yu and Wright, J. Biol. Chem. 271(8): 4110-4105 (1996)] and only occursat high concentrations of LPS [Hailman et al., J. Exp. Med. 179(1):269-277 (1994)]. Binding of LPS to CD14 is greatly accelerated by LBP[Hailman et al. (1994), supra; Tobias et al. (1995), supra; Yu et al.(1996), supra], and LBP is required for activation of cells by eithermCD14 or sCD14 at physiological concentrations of LPS [Schumann et al.(1990), supra; Wright et al. (1990), supra].

LBP is a 60 kD glycoprotein synthesized in the liver and present innormal human serum. LBP belongs to the group of plasma proteins calledacute phase proteins, including C-reactive protein, fibrinogen and serumamyloid A, that increase in concentration in response to infectious,inflammatory and toxic mediators. LBP expression has been induced inanimals by challenge with LPS, silver nitrate, turpentine andCorynebacterium parvum [Geller et al., Arch. Surg. 128(1): 22-28 (1993);Gallay et al., Infect. Immun. 61(2): 378-383 (1993); Tobias et al., J.Exp. Med. 164: 777-793 (1986)]. However, while administration of silvernitrate caused LBP levels to increase in several strains of mice, thiswas not observed in one strain, C3H/HeJ, in which LPS does not induce aninflammatory response [Gallay et al. (1993), supra]. Recently, ananalysis of different human disease states has indicated that increasedLBP levels are uniquely correlated with exposure to LPS. In humanpatients with presumed gram-negative sepsis, serum LBP levels can reachfrom about 50 to about 100 μg/mL [U.S. Pat. No. 5,484,705]. In contrast,in other disease states, such as rheumatoid arthritis, involving anacute phase response in which elevated levels of the acute phaseproteins CRP and fibrinogen were measured in patient serum samples, nosignificant increases in LBP levels were observed. Elevated,particularly persistently elevated, LBP levels have been correlated withpoor clinical outcome in septic patients [U.S. Pat. No. 5,484,705, andU.S. Ser. No. 08/377,391 filed Jan. 24, 1995, both of which are herebyincorporated by reference in their entirety]. This has been confirmed bySchumann et al., 36th Int'l Conf. on Antimicrobial Agents andChemotherapy, New Orleans, La., Sep. 15-18, 1996.

LBP is reported to bind to LPS aggregates (at low LBP to LPS ratios) orto disaggregate LPS vesicles (at high LBP to LPS ratios) [Tobias et al.(1995), supra] to form an LBP:LPS complex that greatly facilitatesbinding of LPS to either mCD14 or sCD14 [Wright et al., J. Exp. Med.173(5): 1281-1286 (1991); Hailman et al. (1994), supra; Yu et al.(1996), supra; Tobias et al. (1995), supra]. LBP is reported to actcatalytically in facilitating LPS binding to CD14, a single LBP moleculeenabling the transfer over 100 LPS molecules to CD14 [Hailman et al.(1994), supra]. LBP is also reported to remain associated with LPSaggregates or LPS coated particles and facilitate binding to cellsexpressing mCD14 in a phenomenon known as opsonization [Wright et al.,J. Exp. Med. 170(4): 1231-1241 (1989); Kirkland et al., J. Biol. Chem.268(33): 24818-24823 (1993); Gegner et al., J. Biol. Chem. 270(10):5320-5325 (1995)]. Thus, LBP potentiates the inflammatory activity ofLPS and is recognized as an immunostimulatory molecule. Functionalanalysis of the LBP molecule has demonstrated that LPS binding residesin the approximate N-terminal half of the protein, but the C-terminalhalf is required to permit transfer of LPS to CD14 [U.S. applicationSer. No. 08/261,660 filed Jun. 17, 1994; Theofan et al., J. Immunol.152(7): 3624-3629 (1994); Han et al., J. Biol Chem. 269(11): 8172-8175(1994)]. Because of the observed potentiating effect LBP has on theinflammatory potential of LPS, blocking or interfering with theimmunostimulatory activity of LBP has been a therapeutic target ofinterest.

For example, a polyclonal antibody preparation to murine LBP has beenshown to prevent LBP mediated binding of LPS to murine macrophages andsubsequent induction of TNF expression in vitro, effectivelyneutralizing the activity of LBP. This same polyclonal antibody was ableto reduce lethality in a murine model of endotoxemia [Gallay et al.(1993), supra].

Several modified forms of LBP have been developed that bind LPS but lackthe ability to transfer the LPS molecule to CD14. U.S. application Ser.No. 08/261,660 filed Jun. 17, 1994, hereby incorporated by reference inits entirety, describes novel biologically active polypeptidederivatives of LBP, including LBP derivative hybrid proteins, which arecharacterized by the ability to bind to LPS and which lack CD14-mediatedimmunostimulatory properties, including the ability of LBP holoproteinto mediate LPS activity via the CD14 receptor. More particularly, theseLBP protein derivatives including LBP derivative hybrid proteins lackingthose carboxy terminal-associated elements characteristic of the LBPholoprotein which enable LBP to bind to and interact with the CD14receptor on monocytes and macrophages so as to provide animmunostimulatory signal to monocytes and macrophages. Such LBP proteinderivatives included those characterized by a molecular weight less thanor equal to about 25 kD, including an amino-terminal LBP fragment havingamino acid residues 1-197 that was designated rLBP₂₅. This recombinantprotein corresponding to the amino-terminal residues 1-197 of LBP hasbeen shown to bind LPS but could neither facilitate binding of LPS toCD14 nor permit LPS-induced expression of TNF [see also, Theofan et al.(1994), supra; Han et al. (1994), supra]. Additionally, this N-terminalfragment was shown to inhibit LPS-induced expression of TNF that wasmediated by full-length LBP [Han et al. (1994), supra]. rLBP₂₅ includesamino acid regions comprising LBP residues 17 through 45, 65 through 99and 141 through 167 which correspond to respective biologically active(e.g., LPS binding) domains (e.g., Domain I—residues 17 through 45;Domain II—residues 65 through 99; and Domain III—residues 142 through169) of bactericidal/permeability-increasing protein (BPI). The LBPderivative hybrid proteins included hybrids of LBP protein sequenceswith the amino acid sequences of other polypeptides and alsocharacterized by the ability to bind to LPS and the absence ofCD14-mediated immunostimulatory properties. Such hybrid proteinsincluded fusions of LBP amino-terminal fragments with polypeptidesequences of other proteins such as BPI, immunoglobulins and the like.Properties of several LBP/BPI fusion proteins have been described byAbrahamson et al., J. Biol. Chem. 272(4):2149-2155 (1997). In addition,a recombinant hybrid fusion between the N-terminal 199 amino acidresidues of LBP and the C-terminal 257 residues of BPI was shown to beprotective in a rodent model of gram-negative sepsis [Opal et al.,Antimicrob. Agents Chemother. 39(12): 2813-2815 (1995)]. U.S.application Ser. No. 08/261,660 filed Jun. 17, 1994 also describes LBPderivatives in the form of synthetic LBP peptides that are portions ofthe LBP sequence corresponding to either Domain II (residues 65-99) orDomain III (residues 142-169) of BPI. The LBP derivative designatedLBP-1 consisted of residues 73 through 99 of LBP. The LBP derivativedesignated LBP-2 consisted of residues 140 through 161 of LBP. Inaddition, Taylor et al., J. Biol. Chem. 270(30): 17934-17938 (1995),described synthetic peptides corresponding to residues 91-105 or 94-108of the mature LBP protein that were reported to compete with LBP forbinding to LPS and could inhibit LPS-induced expression of TNF in vitro.

In addition to transferring LPS to CD14, LBP can facilitate the transferof LPS to serum lipoproteins [Wurfel et al., J. Exp. Med. 180: 1025-1035(1994)]. Association with lipoproteins greatly reduces the inflammatorypotential of LPS [Ulevitch and Johnston (1978), supra]. Thus, LBP itselfcan also participate in the neutraization of LPS. The significance ofLBP-mediated transfer of LPS to lipoproteins, however, remains unclear.Specifically, elevated levels of LBP found in acute phase serum havebeen correlated with a reduction of the rate of association of LPS withlipoproteins [Tobias and Ulevitch, J. Immunol. 131(4): 1913-1916 (1983);Tobias et al. (1985), supra; U.S. Pat. Nos. 5,245,013 and 5,310,879].This ability of LBP to inhibit, rather than facilitate, the transfer ofLPS to lipoproteins was exploited in the initial purification of LBP[Tobias et al. (1986), supra].

Dedrick et al., J. Endotoxin Research 3(supp. 1): 18 (Abstract I-14)(October 1996) reported in an abstract that concentrations of 1 ng/mL to1 μg/mL of rLBP fully potentiated the induction of TNF expression inserum-free medium by 1 ng/mL LPS on a human monocytic cell line (THP.1).In medium containing 10% serum, LBP concentrations of 30 μg/mL orgreater inhibited LPS-induced TNF expression by the THP. 1 cells andalso inhibited E-selection expression in human umbilical endothelialcells (HUVEC) induced by 10 ng/mL LPS. Moreover, it was reported thatadministration of 5 mg/kg rLBP also increased survival in micechallenged with up to 25 mg/kg E. coli LPS. However, human subjectssuffering from disorders involving bacteria and their endotoxin (such assepsis) have been shown to exhibit substantially elevated levels of LBPin circulation (at concentrations of 50 μg/mL to 100 μg/mL of serum),yet these high circulating levels of LBP do not appear to have inhibitedthe adverse effects of bacterial endotoxin in circulation that wereexperienced by these subjects. The role of LBP in promoting oralleviating adverse effects of endotoxin in circulation thus remainsunclear.

Bactericidal/permeability-increasing protein (BPI) is a basic proteinfound in the azurophilic granules of polymorphonuclear leukocytes [Weisset al., J. Biol. Chem. 253(8): 2664-2672 (1978)]. BPI binds to LPS,resulting in its clearance and neutralization. The amino acid sequenceof BPI is closely related to that of LBP [Schumann et al. (1990),supra], and like LBP, the amino-terminal half of BPI has a binding sitefor LPS [Ooi et al., J. Exp. Med. 174: 649-655 (1991)]. However, BPI hasa higher affinity for LPS than does LBP [Gazzano-Santoro et al., Infect.Immun. 62(4): 1185-1191 (1994); Wilde et al., J. Biol Chem. 269(26):17411-17416 (1994)], and cannot transfer LPS to the CD14 molecule. Thus,BPI effectively competes with LBP for LPS binding [Heumann et al., J.Infect. Dis. 167: 1351-1357 (1993); Gazzano-Santoro et al. (1994),supra] and blocks the inflammatory activity of LPS in vitro [Marra etal., J. Immunol 144(2): 662-666 (1990); Ooi et al. (1991), supra], andin humans [de Winter et al., J. Inflamm. 45: 193-206 (1995)].

It has been suggested that sCD14 could be a useful therapeutic agent inendotoxin-related disorders [Schütt et al. (1991), supra; Schütt et al.(1992), supra; Haziot et al. (1994), supra; Haziot et al. (1995),supra]. The presence of sCD14 reduces the amount of 1,LPS complexed withLBP [Tobias et al. (1995), supra], because, although LBP has a higheraffinity for LPS than sCD14, the distribution of LPS between sCD14 andLBP depends on the molar ratio of the two proteins. sCD14 has been shownto inhibit responses that depend on mCD14 [Schütt et al., Res. Immunol143: 71-78 (1992); Schütt et al., Allerg. Immunol. 37: 159-164 (1991);Haziot et al., J. Immunol. 152: 5869-5876 (1994)], and to protect miceagainst experimental endotoxemia [Haziot, et al., J. Immunol. 154:6529-6532 (1995)].

SUMMARY OF THE INVENTION

The present invention is based on the discovery that lipopolysaccharidebinding protein (LBP), an agent previously thought to be stimulatory ofthe adverse effects of bacteria and their endotoxin, can actually reducethe inflammatory potential of bacteria and their endotoxin whenadministered to certain subjects prior to exposure to the bacterialendotoxin. In particular, the invention relates to the administration ofLBP in an amount effective to inhibit the adverse effects of bacterialendotoxin to a subject who has a circulating level of LBP in the normalrange as measured by a quantitative LBP assay. The LBP is administeredat a time prior to exposure to bacterial endotoxin (i.e., when thesubject will be at risk of such exposure). Such times of “at risk ofexposure to endotoxin” include circumstances or conditions associatedwith increased translocation of gut associated bacteria and endotoxin,particularly prior to surgery. Thus, these subjects are administeredprophylactic doses of LBP, to raise the circulating LBP levels totherapeutically effective amounts, in advance of exposure to bacterialendotoxin. The invention thus provides a method of protection againstthe adverse effects of bacterial endotoxin at a time prior to endotoxinchallenge/exposure.

Numerous additional aspects and advantages of the invention will becomeapparent to those skilled in the art upon consideration of the followingdetailed description of the invention which describes presentlypreferred embodiments thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B display the effect of varying concentrations of LBP onLPS-induced TNF expression by monocytic THP. 1 cells in serum-free andserum-containing medium, respectively.

FIGS. 2A and 2B display the effect of varying concentrations of LBP onE-selectin expression by HUVEC induced by LPS and 1L-1β, respectively.

FIGS. 3A, 3B and 3C display the effect of 5 mg/kg LBP on survival ofmice administered lethal doses of 15, 20 and 25 mg/kg LPS, respectively.

DETAILED DESCRIPTION

According to the invention, LBP therapy is specifically useful whenprophylactically administered to subjects who have circulating LBPlevels in the normal range but who are at risk for exposure to bacterialendotoxin. According to the invention, the LBP levels of the subject maybe determined using a sensitive and specific assay, such as described inExample 2 below, and LBP is administered to the subject to increasecirculating LBP levels to a prophylactically/therapeutically effectivelevel, for example, from about 15 to about 100 μg/mL, to prevent/inhibitthe adverse effects of subsequent exposure to bacterial endotoxin.

Since its identification, LBP has been described as an immunostimulatorymolecule, because it potentiates the biological effects ofLPS/endotoxin. However, in vitro experiments have demonstrated that theamount of LBP needed to potentiate LPS-induced expression ofinflammatory molecules was far below that found in normal human serum.In addition, expression of inflammatory molecules induced by bacterialendotoxin was actually inhibited in vitro when the concentration of LBPwas raised above normal circulating concentrations. Furthermore,administration of 5 mg/kg recombinant human LBP to mice following alethal challenge of endotoxin reduced lethality. Thus, these resultssuggested that increased levels of circulating rLBP were protectiveagainst endotoxin. However, circulating LBP levels have been measuredand found to be substantially elevated in human subjects suffering fromsuch disorders involving exposure to bacteria and their endotoxin, suchas sepsis, meningococcemia and confirmed abdominal infections. Despitehaving high circulating levels of LBP (which can rise to greater than100 μg/mL), these subjects still experienced the adverse effects ofbacteria and their endotoxin in circulation.

The present invention is based upon the understanding that increasingLBP levels by administration of LBP protect a subject from the adverseeffects of endotoxin exposure if the circulating LBP level is increasedbefore exposure to endotoxin, rather than after exposure to endotoxin.

The present invention specifically contemplates a method for inhibitingadverse effects of endotoxin in circulation, involving determining thecirculating lipopolysaccharide binding protein (LBP) level of a subjectat risk for exposure to endotoxin, and administering to said subjecthaving a circulating LBP level within the normal range an amount of LBPeffective to elevate the circulating LBP level to inhibit the adverseeffects of exposure to endotoxin, preferably to a level from about 15μg/mL to about 100 μg/mL.

Another aspect of the present invention provides compositions comprisinglipopolysaccharide binding protein (LBP) in a solution buffered at aboutpH 7.5 and containing a poloxamer surfactant, and compositionscomprising LBP in a solution buffered at about pH 7.5 and containing apoloxamer surfactant and a polysorbate surfactant.

As used herein, “circulating LBP level within the normal range,” or“normal circulating LBP level,” means, for humans, LBP concentrations inserum or plasma of from about 1 to about 12 μg/mL as measured by theassay described in Example 2 below, using the rLBP of Example 1 as astandard. The normal range may vary depending on the assay and the LBPstandards utilized, but can be determined for any assay and LBP standardusing a representative population of normal human sera or plasma.

As used herein, “at risk for exposure to endotoxin” includecircumstances or conditions, for example, undergoing surgery or surgicalprocedures including transplantation, that are associated with increasedtranslocation of gut-associated bacteria and their endotoxin.

It is contemplated that polypeptide derivatives of LBP (i.e., fragmentsof LBP, analogs of LBP in which an amino acid has been deleted, insertedor modified, and fusion proteins comprising LBP) that retain the abilityto bind LPS may be administered instead of, or in addition to, LBP, andinclude derivatives that exhibit CD 14-mediated immunostimulatoryproperties. Such derivatives may be obtained by any synthetic orrecombinant means known in the art.

It is contemplated that LBP or derivatives thereof may be administeredaccording to the invention in a pharmaceutical composition withpharmaceutically acceptable diluents, adjuvants, and carriers. Aparticularly preferred composition of rLBP comprises 1-2 mg/mL LBP in 10mM HEPES buffer, 150 mM NaCl, pH 7.5 with 0.2% poloxamer 188 and 0.002%polysorbate 80. A preferred composition of rLBP₂₅ is similar to that forrLBP except that 1.0M NaCl instead of 150 mM NaCl is used. According tothe invention, LBP or derivatives thereof may be administeredsystemically and most preferably intravenously in amounts broadlyranging from about 0.1 mg/kg to about 100 mg/kg of body weight of thetreated subject, preferably at dosages ranging from about 1 mg/kg toabout 25 mg/kg of body weight. Other systemic routes of administrationinclude oral, intravenous, intramuscular or subcutaneous injection(including into a depot for long-term release), intraocular andretrobulbar, intrathecal, intraperitoneal (e.g. by intraperitoneallavage), intrapulmonary (using powdered drug, or an aerosolized ornebulized drug solution), or transdermal. The treating physician mayfind it advantageous to continue the prophylactic treatment bycontinuous infusion or intermittent injection or infusion, at the same,reduced or increased dose per day.

It is further contemplated that LBP or derivatives thereof may beco-administered in conjunction with other agents that bind, clear and/orneutralize endotoxin. “Concurrent administration,” or“co-administration,” as used herein includes administration of multipleagents, in conjunction or combination, together, or before or after eachother. The LBP or derivative and second agent(s) may be administered bydifferent routes, e.g., LBP may be given intravenously while the secondagent(s) is(are) administered intramuscularly. The LBP or derivative andsecond agent(s) may be given sequentially in the same intravenous lineor may be given in different intravenous lines. The agents may beadministered simultaneously or sequentially, as long as they are givenin a manner sufficient to allow all agents to achieve effectiveconcentrations at the site of action.

The present invention further provides a novel use for LBP and fragmentsor derivatives thereof in the manufacture of medicaments forprophylactically treating subjects at risk of exposure to endotoxin.

Example 1 addresses production and purification of rLBP suitable for useas a therapeutic or for use as a standard in the LBP assay of Example 2.Example 2 addresses a sensitive and specific assay for determining LBPlevels in human body fluids. Example 3 addresses in vivo effects of LBP.

EXAMPLE 1 Production and Purification of rLBP

Recombinant LBP (rLBP), suitable for use as a therapeutic according tothe invention or for use as a standard in the assay of Example 2 below,was produced and purified as follows. Plasmid pING4539, containing theDNA encoding full length human LBP (amino acids 1-452, designated“rLBP,” plus the 25 amino acid signal sequence) [SEQ ID NOS: 1 and 2],was prepared as described in Example 2 of U.S. application Ser. No.08/261,660 filed Jun. 17, 1994, hereby incorporated by reference in itsentirety.

CHO-DG44 cells were transfected with linearized pING4539 DNA (40 μg,digested with PvuI, phenol-chloroform extracted and ethanolprecipitated) using electroporation. Following recovery, the cells werediluted and 1×10⁴ cells were plated per 96well plate well in selectivemedium consisting of an αMEM medium lacking nucleosides (IrvineScientific) and supplemented with dialyzed fetal bovine serum (100 mLserum dialyzed against 4 L cold 0.15 NaCl using 6000-8000 cutoff for 16hours at 4° C.). Untransfected CHO-DG44 cells were unable to grow inthis medium because they possess the DHFR⁻ mutation and were removedduring successive feedings with the selective medium. At 1.5-2 weeks,microcolonies consisting of transfected cells were observed.

Clones were analyzed for the presence of LBP-reactive protein in cultureby ELISA in Immulon-II 96 well plates (Dynatech). Supernatant sampleswere added to the plates and incubated 46 hours at 4° C., followed byaddition of goat anti-LBP antiserum and peroxidase-labeled rabbitanti-goat anti-serum. The 21 most productive positive clones wereexpanded in selective αMEM medium and then grown in selective mediumsupplemented with 0.05 μM methotrexate. The best producing amplifiedclone was chosen based on ELISA of supernatants as described above andthen expanded in αMEM media containing 0.05 μM methotrexate for growthin roller bottles.

The transfected CHO-DG44 cells were cultured as follows. All incubationswere performed in a humidified 5% CO₂ incubator maintained at 37° C.Working stock cultures were grown in DME/F-12 with 10% FCS, and afterfour days of growth were seeded into five 2-liter roller bottles. Afteranother six days of incubation, these cells were harvested and seededinto 40 2-liter roller bottles using 1×10⁷ cells in 500 mL of DME/F-12with 5% FCS for each bottle. Four days later, the culture supernatantsfrom each bottle were removed and replaced with 500 mL of fresh DME/F-12with 2.5% FCS, and 10 mL of an S-Sepharose (Pharmacia) ion exchangeresin slurry (50% v/v, sterilized by autoclaving). After four days ofincubation, the media containing S-Sepharose was harvested and replacedwith fresh media again containing S-Sepharose. This process was repeatedone more time, to yield a total of three harvests of S-Sepharose. Eachharvest was processed for purification separately until the LBP waseluted from the S-Sepharose beads, and then the three eluates werepooled for the remainder of the purification procedure. Purificationmethods using S-Sepharose have been described in U.S. Pat. No.5,439,807, hereby incorporated by reference in its entirely.

All chromatographic resins used in the purification of rLBP werepurchased sterile (in ethanol) and equilibrated with pyrogen free bufferor were depyrogenated by immersion in 0.2N NaOH, 1 M NaCl and thenrinsed with pyrogen-free water followed by equilibration with theappropriate buffer. All buffers and reagents were prepared with bottled,pyrogen-free water for irrigation (Baxter).

After the S-Sepharose beads were harvested from the roller bottles, theywere allowed to settle out of the media. The beads were then batchwashed with approximately 800 mL of 20 mM MES, pH 6.8, 150 mM NaCl.After washing with approximately 400 mL of 20 mM sodium acetate (NaOAc),pH 4.0, 150 mM NaCl, the beads were loaded into a 2.5×50 cm column. Thecolumn was washed with 20 mM NaOAc, pH 4.0, 400 mM NaCl, until theabsorbance at 280 nm (A₂₈₀) approached zero, which typically requiresapproximately 600 mL of buffer. The column was then washed with 20 mMNaOAc, pH 4.0, 600 mM NaCl, again until the A₂₈₀ reading returned tozero, which typically requires about 600 mL of buffer. The rLBP waseluted with 20 mM NaOAc, pH 4.0, 1.0 M NaCl. The column was additionallywashed with 20 mM NaOAc, 1.5 M NaOAc, pH 4.0, 1.5 M NaCl to insure allthe protein was eluted. Column fractions containing the protein wereanalyzed by SDS-PAGE, and those containing rLBP were pooled. The pooledfractions were adjusted to a final salt concentration of 200 mM NaClwith 20 mM MES, pH 5.0. This material was then filtered through a 0.2 μmfilter.

The pooled filtrate was applied to a 20 mL Q-Sepharose column forremoval of nucleic acids and then concentrated on a 20 mL S-Sepharosecolumn equilibrated with 20 mM MES, pH 5.0, 200 mM NaCl. The S-Sepharosecolumn was washed with 20 mM MES, pH 5.0, 400 mM NaCl and again with 20mM MES, pH 5.0, 550 mM NaCl, using about 200 mL of buffer per wash. TheLBP was then eluted with 20 mM MES, pH 5.0, 1.2 M NaCl in about 40 mL ofvolume. The LBP was buffer exchanged into 10 mM HEPES, pH 7.5, 150 mMNaCl using a 500 mL Sephacryl S-100 column. Fractions were analyzed withSDS-PAGE, and rLBP-containing fractions were pooled. The final proteinconcentration was adjusted to 1.0 mg/mL and the material was formulatedto 0.2% poloxamer 188 (PLURONIC F-68, BASF Wyandotte Corp., Parsippany,N.J.), 0.002% polysorbate 80 (TWEEN 80, ICI Americas, Inc., Wilmington,Del.). The formulated protein was aliquoted for storage and frozen at−70 degrees until use. The yields from this procedure generally rangefrom about 140 mg to about 300 mg rLBP, typically about 200-240 mg rLBP.

EXAMPLE 2 Determination of LBP Levels in Human Plasma and Sera

The ranges of circulating LBP levels for healthy human subjects and forhuman subjects from a variety of patient populations was determined byassaying representative samples of human plasma or sera with a sandwichELISA.

The LBP assay was validated by evaluating for interference by othercompounds, recovery, precision and clinical sensitivity and specificity.The potential for interference by BPI (which has>45% sequence homologywith LBP), LPS, various blood preservatives commonly used in bloodcollection and heat treatment of blood (to 56° C. or 60° C. toinactivate complement) was investigated. The rLBP prepared as describedin Example 1 was used as a standard; the activity of the formulated LBPwas shown to be constant over time after storage. rBPI and rBPI₂₃ (therecombinant expression product of DNA encoding residues 1 to 199 of BPI)was prepared in the manufacturing facility of XOMA Corporation,Berkeley, Calif. for clinical trials, by a process essentially asdescribed on a smaller scale by Horwitz et al., Protein Expression andPurification 8:28-40 (1996).

Affinity-purified rabbit anti-LBP was prepared by procedures well knownin the art. Briefly, two rabbits were hyper-immunized with LBP, andpooled antisera from these rabbits was diluted with an equal volume ofphosphate buffered saline (PBS), pH 7.2. An LBP-Sepharose column wasprepared by coupling rLBP produced and purified as described in Example1 to cyanogen bromide-activated Sepharose 4B. A portion of the dilutedantisera was passed through the LBP-Sepharose column; the column wasthen washed and bound antibodies were eluted with 0.1M glycine, pH 2.5.Collected fractions were immediately neutralized with 1M sodiumphosphate buffer pH 8.0. Peak fractions were identified by measuringabsorbance at 28 nm. Several sequential column runs were performed andall peak fractions from each column run were pooled. This pool ofaffinity-purified rabbit anti-LBP antibody was assigned a lot number andqualified based on consistent performance in the ELISA.

Biotin-labeled rabbit anti-LBP antibody was prepared by procedures wellknown in the art. Briefly, the antibody was biotin labeled withbiotinamidocaproate N-hydroxysuccinimide ester (Sigma) in 0.1 M sodiumbicarbonate, pH 8.3. Unconjugated biotin was removed and alkaline bufferexchanged by passing the antibody over a PD-10 column (Pharmacia)equilibrated with PBS containing 0.1% sodium azide. This biotin-labeledantibody was assigned a lot number, stored at 2° C. to 8° C., andqualified based on consistent performance in the ELISA.

The sandwich ELISA procedure was carried out as follows. Fifty μL ofaffinity purified rabbit anti-LBP antibody (2 μg/mL in PBS) was added toeach well of Immulon 2 (Dynatech) microtiter plates and incubatedovernight at 2-8° C. The antibody solution was removed and 200 μl of 1%non-fat milk (Carnation or equivalent) in PBS was added to all wells.After blocking the plates for 1 hour at room temperature, the wells werewashed 3 times with 300 μl/well of wash buffer [PBS/0.05% TWEEN 20(polysorbate 20, Sigma, St. Louis). Standards, samples and controls werediluted in triplicate with PBS containing 1% bovine serum albumin, 0.05%TWEEN 20 [PBS-BSA/TWEEN] and 10 units/mL of sodium heparin (Sigma), inseparate 96-well plates. rLBP standard solutions were prepared as serialtwo-fold dilutions of concentrations from 100 to 0.012 ng/mL. Eachreplicate and dilution of the standards, samples and controls (50 μl)was transferred to the blocked microtiter plates and incubated for 1hour at 37° C. After the primary incubation, the wells were washed 3times with 300 μl/well of wash buffer. For each assay, biotin-labeledrabbit anti-LBP antibody was diluted 1/2000 in PBS-TWEEN and 50 μl wasadded to all wells. The plates were then incubated for 1 hour at 37° C.All wells were washed 3 times with 300 μl/well of wash buffer. Alkalinephosphatase-labeled streptavidin (Zymed) was diluted 1/2000 inPBS-BSA/TWEEN and 50 μl was added to all wells. After incubation for 15minutes at 37° C., all wells were washed 3 times with 300 μl/well ofwash buffer) and 3 times with 300 μl/well of deionized water and thesubstrate p-nitrophenylphosphate (1 mg/mL in 10% diethanolamine buffer)was added in a volume of 50 μl to all wells. Color development wasallowed to proceed for 1 hour at room temperature, after which 50 μl of1 N NaOH was added to stop the reaction. The absorbance at 405nm (A₄₀₅)was determined for all wells using a VMAX Plate Reader (MolecularDevices). The mean A₄₀₅ for each set of triplicates was calculated.Outliers (data points deviating more than 20% from the mean) wererejected. A standard curve was plotted at A₄₀₅ versus ng/mL of LBP. Thelinear range was selected, a computerized (RS/1 Release 4.4.4, BoltBeranek and Newman, Inc.) linear curve fit was performed, andconcentrations were determined for samples and controls by interpolationfrom the standard curve. Sample curves are visually compared to thestandard curve to ensure approximately parallel slopes before data areaccepted. Pooled plasma, sera or urine from normal healthy human donorswere used as the appropriate “negative” controls. An Assay ControlSample (ACS), prepared by adding rLBP to normal human plasma to aconcentration of 50,000 ng/mL, was used as a “positive” control.

Western blot analysis was also performed on selected serum and plasmasamples. Briefly, samples were incubated in microtiter wells coated withrabbits anti-LBP that had been prepared and blocked as described abovefor the sandwich ELISA assay. Six replicate samples were incubated withthe rabbit anti-LBP for 1 hour at 37° C. After incubation, LBP waseluted with sample buffer and pooled. Ten μL of eluate from each samplewas run on a non-reducing 10% gel under the conditions of Laemmli,Nature 227:680-685 (1970). Proteins were transferred to nitrocelluloseby standard techniques [Towbin et al., Proc. Nat'l. Acad. Sci.76:4350-4354 (1979)] and probed for immunoreactivity with biotin-labeledrabbit anti-LBP antibody (diluted 1/2000 in 0.25M Tris-HCl, pH 7.2, 0.2MNaCl, 0.3% TWEEN 20) followed by alkaline phosphatase-conjugatedstreptavidin (diluted 1/2000 in the same buffer). Blots were immersed ina 50 μg/mL solution of 5-bromo4chloro3-indolyl phosphate (Sigma) in0.12M veronal-acetate buffer, pH 9.8, containing 0.01% (w/v) nitro bluetetrazolium and 4mM MgCl₂. Color development was allowed to proceed for1 hour at room temperature.

Parameters for the optimization of the LBP sandwich ELISA wereevaluated, including signal to noise ratio, the concentration of rabbitanti-LBP and biotin-labeled rabbit anti-LBP antibodies, and curve fit.The optimal concentrations for the rabbit anti-LBP and biotin-labeledrabbit anti-LBP antibodies used in these experiments were 2 μg/mL and a1:2000 dilution, respectively. The standard curve demonstratedreproducibility with a consistent slope and acceptable signal to noiseratio (>10:1). The linear range for the standard curve was 164-781pg/mL.

For assay validation, the LBP sandwich ELISA was characterized bydetermining assay precision, recovery and the least detectableconcentration of rLBP. These parameters were investigated by performingthe sandwich ELISA on human plasma spiked with different concentrationsof rLBP and then frozen and thawed to mimic the processing of clinicalsamples.

Assay precision was expressed as the coefficient of variation for LBPvalues of the assay control sample (ACS) and normal human plasma (NHP)measured more than 40 times. The mean and standard deviation for ACS andNHP were 39,300±7,930 ng/mL and 2,970±349 ng/mL, respectively. The CVfor the ACS and NHP were 20% and 12%, respectively. A more recentevaluation of assay precision produced comparable values for ACS and NHPof 43,220±8543 ng/mL (CV 19.8%) and 4,372±315 ng/mL (CV 7.2%),respectively. The NHP values are consistent with the endogenous levelsof LBP in normal human sera reported by Leturcq et al., J. Cell.Biochem., Suppl. 16C:161 (1992) which ranged from 1 to 24 μg/mL, with anaverage of 7 μg/mL. Acceptance of assay data is based on the ACS valuesobtained with each assay.

Average recovery of LBP spiked into human plasma (defined as the amountof rLBP measured in spiked samples minus the concentration in theunspiked control, divided by the actual amount spiked in the sample) was68% across a rLBP concentration range of 0 to 168,000 ng/mL. Averagerecovery of LBP spiked into human urine was 77% over an rLBP range of 3to 1000 ng/mL. The lowest detectable concentration of LBP spiked intohuman plasma (producing a discernible signal above background) was 0.164ng/mL.

Western blot data clearly demonstrated that the sandwich ELISA isspecific for LBP. When plasma samples from normal and presumed septicpatients were evaluated, all patient samples exhibited a single 60 kDband similar to the LBP controls. In the sandwich ELISA, LBP levels forthese patients ranged from 3.35 to 113 μg/mL.

The sandwich ELISA was also performed on blood samples that had beenheat-treated to 56° C. or 60° C. Heat treatment diminished the amount ofLBP detected (compared to sera maintained at 4° C.) by 34% and 97%,respectively.

For the interference studies, the sandwich ELISA and Western blotanalysis were performed on donor blood samples collected in tubescontaining acid-citrate-dextrose (ACD), ethylene-diaminetetraacetic acid(EDTA) or heparin, and on control samples of donor blood collectedwithout preservatives and control samples of commercially availablenormal human serum. Blood preservatives did not interfere with thedetection of LBP using the sandwich ELISA. Results obtained using thesame blood collected with and without preservatives were comparable.Western blot analysis revealed a band pattern that was the same as thepattern for the LBP controls or for commercially available normal humansera.

The sandwich ELISA and Western blot analysis were also performed onsamples spiked with varying concentrations of rBPI and rBPI₂₃. Becauseheparin minimizes non-specific adsorption of BPI to the microtiter plate(see U.S. Pat. Nos. 5,466,580 and 5,466,581, both of which are herebyincorporated by reference in their entirety), heparin was added to assaydiluents in order to minimize the signal generated by BPI in thesandwich ELISA. The results of these analyses showed that this sandwichELISA does not demonstrate significant cross-reactivity with BPI. Theaffinity purified rabbit anti-LBP was not cross-reactive with eitherrBPI or rBPI₂₃ on Western blot analysis. In the sandwich ELISA, thereactivity of BPI and rBPI₂₃ at concentrations ranging from 0.78 to 100ng/mL was comparable to background level, but higher concentrations ofboth forms of BPI (greater than 100 ng/mL) demonstrated somecross-reactivity.

In addition, the ability of LPS to interfere with detection of LBP inthe sandwich ELISA was evaluated. No interference by LPS was observed;addition of LPS (at concentrations ranging from 0 to 100 ng/mL) tosamples spiked with varying concentrations (7, 16 and 168 μg/mL) of LBPdid not diminish the amount of LBP detected in the ELISA.

These data confirm that the LBP sandwich ELISA described is specific forLBP. The standard curve for the assay is consistent and reproducible,and the assay can detect concentrations of LBP as low as 164 pg/mL.

This assay was used to determine LBP levels in normal human subjects andin a variety of patient populations. See U.S. Pat. No. 5,484,705 andU.S. Ser. No. 08/377,391 filed Jan. 24, 1995, both of which are herebyincorporated by reference in their entirety. Plasma or sera samples fromhealthy human subjects and humans from a variety of patient populations,including rheumatoid arthritis (RA), acute graft vs. host disease afterbone marrow transplantation (BM aGvHD), acute lymphocytic leukemia(ALL), chronic lymphocytic leukemia (CLL), cutaneous T-cell lymphoma(CTCL), diabetes, psoriasis, scleroderma, sepsis, abdominal infection,meningococcemia, Crohn's disease, aplastic anemia (AA), systemic lupuserythematosus (SLE), acute immunodeficiency syndrome (AIDS), alcoholicfatty liver, alcoholic hepatitis, alcoholic cirrhosis, ulcerativecolitis and non-alcoholic cirrhosis, were assayed as described above.Results are shown below in Table 1. Results for each of the 59 samplesfrom the healthy human population are displayed in Table 2 below. Ofthese 59 samples, 58 samples had LBP levels ranging from 1.43 μg/mL to7.70 μg/mL, while one sample had an LBP level of 11.20 μg/mL.

TABLE 1 Mean μg/mL No. of LBP in Plasma Std. Std. POPULATION Subjects orSera Dev. Error HEALTHY 59 4.1 1.7 0.2 SEPSIS 390 30.6 17.6 0.9ABDOMINAL INFECTION 16 52.1 23.9 6.2 MENINGOCOCCEMIA 17 19.7 9.5 2.4 RA86 7.8 4.8 0.5 BM aGvHD 8 9.1 5.6 2.0 ALL 6 8.7 8.1 3.3 CLL 9 7.7 5.11.7 CTCL 12 7.5 4.2 1.2 DIABETES 13 2.8 1.2 0.3 PSORIASIS 13 8.2 4.0 1.1SCLERODERMA 4 7.2 2.9 1.5 CROHN'S 19 16.1 10.2 2.4 APLASTIC ANEMIA 169.5 9.2 2.3 SLE 10 6.7 2.4 0.8 AIDS 15 5.3 2.3 0.6 ALCOHOLIC FATTY LIVER10 8.5 7.0 2.2 ALCOHOLIC HEPATITIS 9 8.0 4.8 1.6 ALCOHOLIC CIRRHOSIS 1110.9 4.6 1.4 ULCERATIVE COLITIS 7 21.0 19.8 7.5 NON-ALCOHOLIC 6 10.7 8.43.4 CIRRHOSIS

TABLE 2 LBP Levels in Plasma/Sera Samples of 59 Healthy Human Subjectsμg/mL LBP μg/mL LBP μg/mL LBP μg/mL LBP 2.59 5.09 4.57 3.50 2.75 5.114.13 6.40 2.86 5.77 4.11 7.70 2.93 5.95 4.00 3.60 3.19 5.99 4.09 3.603.26 7.41 1.52 1.70 3.64 11.20 3.01 3.40 3.67 3.70 2.93 4.00 3.95 2.013.62 3.50 3.96 1.99 6.20 2.40 4.37 1.43 2.78 6.60 4.41 2.78 3.60 5.604.42 3.62 4.20 5.30 4.91 2.35 4.10 4.40 4.98 2.94 3.20

EXAMPLE 3 In Vivo Effect of LBP

The following is an exemplary procedure for administration of LBPaccording to the invention. A human subject, for example, a patientabout to undergo a surgical procedure, is identified as a subject atrisk for exposure to endotoxin. An LBP assay such as the assay describedabove in Example 2 is performed on plasma or serum from the subject todetermine the subject's circulating LBP level. If the subject'scirculating LBP level is normal, then the subject is treated with an LBPcomposition such as that described above in Example 1 at an appropriatetime before surgery as determined by the treating physician, e.g., from0-24 hours prior to surgery, preferably prior to induction ofanesthesia, and more preferably from 1-2 hours prior to surgery. The LBPcomposition is administered parenterally, e.g., intravenously, and thecirculating prophylactic/therapeutic LBP level after such administrationmay be confirmed by a second LBP assay on plasma or serum from thepatient.

In a separate, related experiment, the effect of LBP on LPS-induced TNFExpression in a monocytic cell line (THP. 1) was evaluated as follows.THP. 1 cells were maintained in RPMI'(GibcoBRL, Gaithersburg, Md.) with10% fetal bovine serum (FBS) and were cultured in RPMI' with 10% FBSplus 50 ng/mL 1,25-dihydroxy-vitamin D (BIOMOL Research LaboratoriesInc, Plymouth Meeting, Pa.) for three days prior to treatment with LPSto induce CD14 expression. Before incubation with LPS, cells were washedthree times with RPMI and suspended in either RPMI with 10% FBS or inserum free medium [RPMI supplemented with 1% HB101 (Irvine Scientific,Santa Ana, Calif.)]. Cells (5×10⁴/well) were added to 96 well plates.Aliquots of rLBP, diluted in the same medium as the cells, were added tofinal concentrations of from 0 to 100,000 ng/mL. Expression of TNF wasinduced by the addition of E. coli O128 LPS (Sigma, St. Louis, Mo.) to afinal concentration of 1 ng/mL. Plates were incubated for 3 hours at 37°C. in 5% CO₂, then an aliquot of the supernatant was removed and assayedfor TNF by the WEHI 164 toxicity assay using CellTiter 96™ AQ_(ueous)(Promega Corp., Madison, Wis.) to monitor cell viability. Resultsdisplayed in FIG. 1A showed that mCD14-dependent induction of TNFexpression by 1 ng/mL LPS in THP.1 cells in serum-free medium waspotentiated by low levels of rLBP (<1 μg/mL) but was inhibited by highlevels of rLBP (>10 μg/mL) in vitro. Results displayed in FIG. 1B showedthat the LPS-induced TNF expression in medium with 10% serum wasinhibited by 30 μg/mL amd 100 μg/mL rLBP.

In another separate, related experiment, the effect of LBP on E-selectinexpression in human umbilical vein endothelial cells (HUVEC) was alsoevaluated as follows. HUVEC (Clonetics, San Diego, Calif.) weremaintained in endothelial cell growth medium (EGM, Clonetics) with 5%FBS. Cells were grown to confluence in 96 well plates, washed, andmedium containing rLBP was added to final concentrations of from 0 to 50μg/mL rLBP. The cells were incubated with 10 ng/mL E. coli O128 LPS or10 pg/mL IL-1β(Genzyme) for 4 hours in M199 (GibcoBRL) with 10% FBS.Cell surface E-selectin expression was measured by ELISA as described inHuang et al., Inflammation 19:389-404 (1995), and is displayed in FIGS.2A and 2B. High levels of rLBP inhibited sCD14-dependent induction ofE-selectin by LPS, but not by 1L-1β.

In yet a further separate, related experiment, the effect of LBPinjected following endotoxin challenge on survival in an mouse model oflethal endotoxemia was evaluated as follows. CD1 mice (n=15 per group).were challenged intravenously with 15, 20, 25 mg/kg E. coli O111:B4 LPS(Sigma) and then immediately treated intravenously with 5 mg/kg rLBP orvehicle (rLBP formulation buffer). Mortality was recorded for 7 days andis displayed in FIGS. 3A, 3B and 3C. The results show thatadministration of 5 mg/kg rLBP to these mice was protective followinglethal endotoxin doses of 15 and 20 mg/kg (p<0.05 vs. LPS alone).

Numerous modifications and variations in the practice of the inventionare expected to occur to those skilled in the art upon consideration ofthe foregoing description of the presently preferred embodimentsthereof. Consequently, the only limitations which should be placed uponthe scope of the present invention are those which appear in theappended claims.

2 1443 base pairs nucleic acid single linear DNA not provided CDS1..1443 mat_peptide 76..1443 misc_feature “rLBP” 1 ATG GGG GCC TTG GCCAGA GCC CTG CCG TCC ATA CTG CTG GCA TTG CTG 48 Met Gly Ala Leu Ala ArgAla Leu Pro Ser Ile Leu Leu Ala Leu Leu -25 -20 -15 -10 CTT ACG TCC ACCCCA GAG GCT CTG GGT GCC AAC CCC GGC TTG GTC GCC 96 Leu Thr Ser Thr ProGlu Ala Leu Gly Ala Asn Pro Gly Leu Val Ala -5 1 5 AGG ATC ACC GAC AAGGGA CTG CAG TAT GCG GCC CAG GAG GGG CTA TTG 144 Arg Ile Thr Asp Lys GlyLeu Gln Tyr Ala Ala Gln Glu Gly Leu Leu 10 15 20 GCT CTG CAG AGT GAG CTGCTC AGG ATC ACG CTG CCT GAC TTC ACC GGG 192 Ala Leu Gln Ser Glu Leu LeuArg Ile Thr Leu Pro Asp Phe Thr Gly 25 30 35 GAC TTG AGG ATC CCC CAC GTCGGC CGT GGG CGC TAT GAG TTC CAC AGC 240 Asp Leu Arg Ile Pro His Val GlyArg Gly Arg Tyr Glu Phe His Ser 40 45 50 55 CTG AAC ATC CAC AGC TGT GAGCTG CTT CAC TCT GCG CTG AGG CCT GTC 288 Leu Asn Ile His Ser Cys Glu LeuLeu His Ser Ala Leu Arg Pro Val 60 65 70 CCT GGC CAG GGC CTG AGT CTC AGCATC TCC GAC TCC TCC ATC CGG GTC 336 Pro Gly Gln Gly Leu Ser Leu Ser IleSer Asp Ser Ser Ile Arg Val 75 80 85 CAG GGC AGG TGG AAG GTG CGC AAG TCATTC TTC AAA CTA CAG GGC TCC 384 Gln Gly Arg Trp Lys Val Arg Lys Ser PhePhe Lys Leu Gln Gly Ser 90 95 100 TTT GAT GTC AGT GTC AAG GGC ATC AGCATT TCG GTC AAC CTC CTG TTG 432 Phe Asp Val Ser Val Lys Gly Ile Ser IleSer Val Asn Leu Leu Leu 105 110 115 GGC AGC GAG TCC TCC GGG AGG CCC ACAGTT ACT GCC TCC AGC TGC AGC 480 Gly Ser Glu Ser Ser Gly Arg Pro Thr ValThr Ala Ser Ser Cys Ser 120 125 130 135 AGT GAC ATC GCT GAC GTG GAG GTGGAC ATG TCG GGA GAC TTG GGG TGG 528 Ser Asp Ile Ala Asp Val Glu Val AspMet Ser Gly Asp Leu Gly Trp 140 145 150 CTG TTG AAC CTC TTC CAC AAC CAGATT GAG TCC AAG TTC CAG AAA GTA 576 Leu Leu Asn Leu Phe His Asn Gln IleGlu Ser Lys Phe Gln Lys Val 155 160 165 CTG GAG AGC AGG ATT TGC GAA ATGATC CAG AAA TCG GTG TCC TCC GAT 624 Leu Glu Ser Arg Ile Cys Glu Met IleGln Lys Ser Val Ser Ser Asp 170 175 180 CTA CAG CCT TAT CTC CAA ACT CTGCCA GTT ACA ACA GAG ATT GAC AGT 672 Leu Gln Pro Tyr Leu Gln Thr Leu ProVal Thr Thr Glu Ile Asp Ser 185 190 195 TTC GCC GAC ATT GAT TAT AGC TTAGTG GAA GCC CCT CGG GCA ACA GCC 720 Phe Ala Asp Ile Asp Tyr Ser Leu ValGlu Ala Pro Arg Ala Thr Ala 200 205 210 215 CAG ATG CTG GAG GTG ATG TTTAAG GGT GAA ATC TTT CAT CGT AAC CAC 768 Gln Met Leu Glu Val Met Phe LysGly Glu Ile Phe His Arg Asn His 220 225 230 CGT TCT CCA GTT ACC CTC CTTGCT GCA GTC ATG AGC CTT CCT GAG GAA 816 Arg Ser Pro Val Thr Leu Leu AlaAla Val Met Ser Leu Pro Glu Glu 235 240 245 CAC AAC AAA ATG GTC TAC TTTGCC ATC TCG GAT TAT GTC TTC AAC ACG 864 His Asn Lys Met Val Tyr Phe AlaIle Ser Asp Tyr Val Phe Asn Thr 250 255 260 GCC AGC CTG GTT TAT CAT GAGGAA GGA TAT CTG AAC TTC TCC ATC ACA 912 Ala Ser Leu Val Tyr His Glu GluGly Tyr Leu Asn Phe Ser Ile Thr 265 270 275 GAT GAG ATG ATA CCG CCT GACTCT AAT ATC CGA CTG ACC ACC AAG TCC 960 Asp Glu Met Ile Pro Pro Asp SerAsn Ile Arg Leu Thr Thr Lys Ser 280 285 290 295 TTC CGA CCC TTC GTC CCACGG TTA GCC AGG CTC TAC CCC AAC ATG AAC 1008 Phe Arg Pro Phe Val Pro ArgLeu Ala Arg Leu Tyr Pro Asn Met Asn 300 305 310 CTG GAA CTC CAG GGA TCAGTG CCC TCT GCT CCG CTC CTG AAC TTC AGC 1056 Leu Glu Leu Gln Gly Ser ValPro Ser Ala Pro Leu Leu Asn Phe Ser 315 320 325 CCT GGG AAT CTG TCT GTGGAC CCC TAT ATG GAG ATA GAT GCC TTT GTG 1104 Pro Gly Asn Leu Ser Val AspPro Tyr Met Glu Ile Asp Ala Phe Val 330 335 340 CTC CTG CCC AGC TCC AGCAAG GAG CCT GTC TTC CGG CTC AGT GTG GCC 1152 Leu Leu Pro Ser Ser Ser LysGlu Pro Val Phe Arg Leu Ser Val Ala 345 350 355 ACT AAT GTG TCC GCC ACCTTG ACC TTC AAT ACC AGC AAG ATC ACT GGG 1200 Thr Asn Val Ser Ala Thr LeuThr Phe Asn Thr Ser Lys Ile Thr Gly 360 365 370 375 TTC CTG AAG CCA GGAAAG GTA AAA GTG GAA CTG AAA GAA TCC AAA GTT 1248 Phe Leu Lys Pro Gly LysVal Lys Val Glu Leu Lys Glu Ser Lys Val 380 385 390 GGA CTA TTC AAT GCAGAG CTG TTG GAA GCG CTC CTC AAC TAT TAC ATC 1296 Gly Leu Phe Asn Ala GluLeu Leu Glu Ala Leu Leu Asn Tyr Tyr Ile 395 400 405 CTT AAC ACC TTC TACCCC AAG TTC AAT GAT AAG TTG GCC GAA GGC TTC 1344 Leu Asn Thr Phe Tyr ProLys Phe Asn Asp Lys Leu Ala Glu Gly Phe 410 415 420 CCC CTT CCT CTG CTGAAG CGT GTT CAG CTC TAC GAC CTT GGG CTG CAG 1392 Pro Leu Pro Leu Leu LysArg Val Gln Leu Tyr Asp Leu Gly Leu Gln 425 430 435 ATC CAT AAG GAC TTCCTG TTC TTG GGT GCC AAT GTC CAA TAC ATG AGA 1440 Ile His Lys Asp Phe LeuPhe Leu Gly Ala Asn Val Gln Tyr Met Arg 440 445 450 455 GTT 1443 Val 481amino acids amino acid linear protein not provided misc_feature “rLBP” 2Met Gly Ala Leu Ala Arg Ala Leu Pro Ser Ile Leu Leu Ala Leu Leu -25 -20-15 -10 Leu Thr Ser Thr Pro Glu Ala Leu Gly Ala Asn Pro Gly Leu Val Ala-5 1 5 Arg Ile Thr Asp Lys Gly Leu Gln Tyr Ala Ala Gln Glu Gly Leu Leu10 15 20 Ala Leu Gln Ser Glu Leu Leu Arg Ile Thr Leu Pro Asp Phe Thr Gly25 30 35 Asp Leu Arg Ile Pro His Val Gly Arg Gly Arg Tyr Glu Phe His Ser40 45 50 55 Leu Asn Ile His Ser Cys Glu Leu Leu His Ser Ala Leu Arg ProVal 60 65 70 Pro Gly Gln Gly Leu Ser Leu Ser Ile Ser Asp Ser Ser Ile ArgVal 75 80 85 Gln Gly Arg Trp Lys Val Arg Lys Ser Phe Phe Lys Leu Gln GlySer 90 95 100 Phe Asp Val Ser Val Lys Gly Ile Ser Ile Ser Val Asn LeuLeu Leu 105 110 115 Gly Ser Glu Ser Ser Gly Arg Pro Thr Val Thr Ala SerSer Cys Ser 120 125 130 135 Ser Asp Ile Ala Asp Val Glu Val Asp Met SerGly Asp Leu Gly Trp 140 145 150 Leu Leu Asn Leu Phe His Asn Gln Ile GluSer Lys Phe Gln Lys Val 155 160 165 Leu Glu Ser Arg Ile Cys Glu Met IleGln Lys Ser Val Ser Ser Asp 170 175 180 Leu Gln Pro Tyr Leu Gln Thr LeuPro Val Thr Thr Glu Ile Asp Ser 185 190 195 Phe Ala Asp Ile Asp Tyr SerLeu Val Glu Ala Pro Arg Ala Thr Ala 200 205 210 215 Gln Met Leu Glu ValMet Phe Lys Gly Glu Ile Phe His Arg Asn His 220 225 230 Arg Ser Pro ValThr Leu Leu Ala Ala Val Met Ser Leu Pro Glu Glu 235 240 245 His Asn LysMet Val Tyr Phe Ala Ile Ser Asp Tyr Val Phe Asn Thr 250 255 260 Ala SerLeu Val Tyr His Glu Glu Gly Tyr Leu Asn Phe Ser Ile Thr 265 270 275 AspGlu Met Ile Pro Pro Asp Ser Asn Ile Arg Leu Thr Thr Lys Ser 280 285 290295 Phe Arg Pro Phe Val Pro Arg Leu Ala Arg Leu Tyr Pro Asn Met Asn 300305 310 Leu Glu Leu Gln Gly Ser Val Pro Ser Ala Pro Leu Leu Asn Phe Ser315 320 325 Pro Gly Asn Leu Ser Val Asp Pro Tyr Met Glu Ile Asp Ala PheVal 330 335 340 Leu Leu Pro Ser Ser Ser Lys Glu Pro Val Phe Arg Leu SerVal Ala 345 350 355 Thr Asn Val Ser Ala Thr Leu Thr Phe Asn Thr Ser LysIle Thr Gly 360 365 370 375 Phe Leu Lys Pro Gly Lys Val Lys Val Glu LeuLys Glu Ser Lys Val 380 385 390 Gly Leu Phe Asn Ala Glu Leu Leu Glu AlaLeu Leu Asn Tyr Tyr Ile 395 400 405 Leu Asn Thr Phe Tyr Pro Lys Phe AsnAsp Lys Leu Ala Glu Gly Phe 410 415 420 Pro Leu Pro Leu Leu Lys Arg ValGln Leu Tyr Asp Leu Gly Leu Gln 425 430 435 Ile His Lys Asp Phe Leu PheLeu Gly Ala Asn Val Gln Tyr Met Arg 440 445 450 455 Val

What is claimed is:
 1. A method for inhibiting adverse effects ofendotoxin in circulation comprising the step of before exposure of asubject to endotoxin, administering to said subject having a circulatingLBP level within the normal range an amount of LBP effective to elevatethe circulating LBP level to inhibit the adverse effects of exposure toendotoxin.
 2. The method of claim 1 wherein the circulating LBP level ofsaid subject is elevated to a level from about 15 μg/mL to about 100μg/mL.
 3. The method of claim 1 wherein the circulating LBP level ofsaid subject continues to be elevated at the time of exposure toendotoxin.
 4. The method of claim 1 further comprising the step ofassaying the circulating LBP level of said subject prior toadministering LBP.
 5. The method of claim 1 further comprising the stepof assaying the circulating LBP level of said subject afteradministering LBP.
 6. The method of claim 1 wherein the subject isundergoing surgery or surgical procedures.
 7. The method of claim 6wherein the subject is undergoing transplantation.
 8. The method ofclaim 6 wherein the LBP is administered from about 0 to 24 hours priorto surgery or surgical procedure.
 9. The method of claim 6 wherein theLBP is administered prior to induction of anesthesia.
 10. The method ofclaim 6 wherein the LBP is administered from about 1 to 2 hours prior tosurgery or surgical procedure.
 11. The method of claim 1 wherein the LBPis administered systemically.
 12. The method of claim 11 wherein the LBPis administered intravenously.